Most of the current drugs in use for the treatment of AIDS work by targeting the enzymatic activities of :he HIV reverse transcriptase or protease, although presently there is also active development of several other targets. However, because the emergence of drug resistant virus commonly occurs as the result of reatment, there remains a great need to continue the search for alternative therapies that target other essential novel viral activities. HIV Rev function represents a heretofore under-exploited anti-viral target. To find Rev inhibitors, we recently designed and performed a screen of 40,000 compounds. As a result of this work, we have identified eight chemically unrelated heterocyclic compounds that appear to inhibit HIV replication and Rev function at uM concentrations of compound. We now propose to further characterize these small molecule inhibitors, focusingon 3 or 4 of the 8 compounds that our preliminary data suggest are the most promising. We will critically evaluate each compound with respect to its modality of action and ability to target a wide range of different HIV isolates. Our main goal is to unequivocally determine if any of these compounds target a specific step in the Rev/RRE pathway, and to define their inhibitory profile on a wide range of HIV isolates. This data will allow us to judge if further pursuit of these compounds, as lead drug candidates, is warranted. However, even if these compounds turn out to be unsuitable for drug development because of potency, toxicity or other unforeseen issues, they may provide important laboratory tools for continued studies on Rev function. Furthermore, during the course of this work, assays will be developed that would be readily adaptable for screening of other candidate Rev inhibitors. Specific Aim #1: To assess the ability of the compounds to inhibit replication of a broad range of HIV-1 isolates, and to compare their effects on Rev function from the various HIV-1 groups and clades. Specific Aim #2: To utilize a variety of cell-based and biochemical assays to determine the precise step in the Rev pathway where the compounds act. Specific Aim #3: To explore the development of resistance to selected Rev inhibitors, to characterize the basis of resistance and to address the issue of cross-resistance.